1. Field of the Invention
The present invention relates to oligonucleotides which hybridize to a region of ribosomal RNA (rRNA), particularly to such oligonucleotides which inhibit protein synthesis.
2. Background of the Invention
DNA oligonucleotides that are complementary to specific messenger RNAs (mRNA) have been used to inhibit synthesis of cellular and viral proteins. These complementary oligonucleotides, commonly known as antisense oligonucleotides, inhibit the translation of specific mRNAs by annealing to the mRNA and preventing formation of the directed protein.
Though there are many examples in the literature of oligonucleotides complementary to mRNAs, reports of the use of oligonucleotides complementary to specific ribosomal RNA (rRNA) sequences to affect protein synthesis are limited. Jayaraman et al. (Proc. Natl. Acad. Sci. USA, 78: 1537-1541, (1981)) have shown the inhibition of bacterial protein synthesis with oligonucleotides complementary to a region of 16S rRNA. However in their paper only a limited number of their complementary oligonucleotides were able to inhibit protein synthesis. Of those affecting protein synthesis only one showed any ability to affect transformed human cells and that oligonucleotide was only able to reduce protein synthesis by 10%.
.alpha.-Sarcin is a toxin that attacks the .alpha.-sarcin recognition domain in 28S rRNA. The .alpha.-sarcin recognition domain is a 14 nucleotide region found, for example, in yeast (Georgier et al., Nucleic Acids Res. 9, 6935-6952, (1981)), Xenopus (Ware et al., Nucleic Acids Res. 11, 7795-7817, (1983)) and rat (Chan et al., Nucleic Acids Res. 11, 7819-7831, (1983)). It is part of a putative 17 nucleotide loop approximately 400 nucleotides from the 3' end of the 28S rRNA (Clark et al., Nucleic Acids Res., 12, 6197-6220, (1984)).
This universally conserved loop of the .alpha.-sarcin domain is accessible to elongation factors EF-G and EF-Tu (MoaZed et al., Nature 334, 334-362, (1988)) as well as a group of toxins. Besides .alpha.-sarcin, this group of toxins includes ricin, Shiga toxin and Shiga-like toxin II which are specific N-glycosidases for both deproteinized 28S rRNA and 28S rRNA in isolated rat ribosomes. These toxins specifically remove the adenosine adjacent to the .alpha.-sarcin cut site (Endo et al., J. Biol. Chem. 262, 8128-8130, (1987); and Endo et al., Eur. J. Biochem. 171, 45-50, (1988)) and thereby inhibit protein synthesis (Ackerman et al., J. Biol. Chem. 263, 17076-17083, (1988) and Saxena st al., J. Biol. Chem. 264, 596-601, (1989)).
Toxin specificity and sites of nucleotide cleavage in the .alpha.-sarcin recognition domain loop of 28S rRNA in living cells was determined by the present inventors (Ackerman et al., J. of Biol. Chem. 263, 17076-17083, (1988) and Saxena et al., J. of Biol. Chem. 264, 596-601, (1989)). It was shown that the toxins .alpha.-sarcin, ricin, Shiga toxin and Shiga-like toxin inhibit protein synthesis in intact Xenopus oocytes by attacking specific nucleotides within the .alpha.-sarcin loop in 28S rRNA. .alpha.-Sarcin specifically cuts 28S rRNA 377 nucleotides from its 3' end; and ricin, Shiga toxin and Shiga-like toxin specifically remove adenine-3732 which is located 378 nucleotides from the 3' end of 28S rRNA.
Henderson and Lake (Proceedings of the 16th FEBS Congress, Part B, VNU Science Press, 219-228, (1985)) reported 50S ribosomal subunit collapse initiated by hybridization of a 14-base cDNA probe to the .alpha.-sarcin domain of Escherichia coli (E. coli) 23S rRNA. Using the same binding buffer and a synthetic 14-mer, White et al. (Nucleic Acids Res. 16, 10817-10831, (1988)) Were unable to hybridize the probe to the .alpha.-sarcin domain of intact 50S subunits. Furthermore, White et al. showed that subunit collapse was induced by binding buffer lacking the oligonucleotide. Thus attempts at simulating toxin inhibition of protein synthesis using oligonucleotides have been unsuccessful.